Glycogen storage disease type I (GSD-I) is a group of autosomal recessive disorders caused by deficiencies in the endoplasmic reticulum (ER)-bound glucose-6-phosphatase (G6Pase) system, a key enzyme complex in glucose homeostasis. The G6Pase system comprises of at least two integral membrane proteins, G6Pase and glucose-6-phosphate transporter (G6PT). G6PT translocates glucose-6-phosphate (G6P), the product of gluconeogenesis and glycogenolysis, from cytoplasm to the lumen of the ER and inside the ER G6Pase catalyzes the hydrolysis of G6P to produce glucose and phosphate. Deficiencies in G6Pase and G6PT cause GSD-1a and GSD-1b, respectively. Both patients manifest the symptoms of G6Pase enzyme deficiency characterized by growth retardation, hypoglycemia, hepatomegaly, nephromegaly, hyperlipidemia, hyperuricemia, and lactic acidemia. However, GSD-Ib patients also present with unique symptoms not so obviously associated with G6Pase activity, including chronic neutropenia and myeloid dysfunctions, which result in recurrent bacterial infections. Over the last 20 years dietary therapy has alleviated some, but not all, metabolic abnormalities of GSD-1 patients and delayed disease progress. However, the underlying disease remains untreated and the efficacy of dietary treatment is frequently limited by poor compliance. Therefore, long-term complications still develop in adult patients. An understanding of the molecular genetics and pathogenesis of GSD-1 is needed to lead to therapies that can rectify the long-term complications of GSD-1. The most prevalent from of GSD-Ia is caused by a deficiency in G6Pase, a key enzyme in glucose homeostasis, catalyzing the hydrolysis of G6P to glucose and phosphate in the terminal steps of gluconeogenesis and glycogenolysis. G6Pase is a highly hydrophobic protein anchored to the ER by 9-transmembrane helices. The protein can not be expressed in a soluble form and must embed correctly in the ER membrane and couple with other proteins to be functional. Therefore, enzyme replacement therapy is not an option, but somatic gene therapy, targeting G6Pase to the liver and the kidney, is an attractive possibility. To develop novel therapies for GSD-Ia, we had previously generated G6Pase-deficient mice that manifest a metabolic profile and phenotype virtually identical to that of human GSD-Ia patients. Using neonatal GSD-Ia mice we now demonstrate that a combined adeno and adeno-associated virus vector-mediated gene transfer leads to sustained G6Pase expression in both the liver and the kidney and corrects the murine GSD-Ia disease for at least 12 months. Our results suggest that human GSD-Ia would be treatable by gene therapy. The amino acids comprising the catalytic center of G6Pase include Lys76, Arg83, His119, Arg170, and His176. During catalysis, a His residue in G6Pase becomes phosphorylated generating an enzyme-phosphate intermediate. It was predicted that His176 would be the amino acid that acts as a nucleophile forming a phosphohistidine-enzyme intermediate and His119 would be the amino acid that provides the proton needed to liberate the glucose moiety. However, the phosphate acceptor in G6Pase has eluded molecular characterization. To identify the His residue that covalently bound the phosphate moiety, we generated recombinant adenoviruses carrying G6Pase wild type and active site mutants. A 40-kDa [32P]-phosphate-G6Pase intermediate was identified after incubating [32P]-glucose-6-phosphate with microsomes expressing wild type but not with microsomes expressing either H119A or H176A mutant G6Pase. Human G6Pase contains five methionine residues at positions, 1, 5, 121, 130, and 279. After cyanogen bromide cleavage, His119 is predicted to be within a 116-amino-acid peptide of 13.5 kDa with an isoelectric point of 5.3 (residues 6-121) and His176 within a 149-amino-acid peptide of 16.8 kDa with an isoelectric point of 9.3 (residues 131-279). We show that after digestion of a non-glycosylated [32P]-phosphate-G6Pase intermediate by cyanogen bromide, the [32P]-phosphate remains bound to a peptide of 17 kDa with an isoelectric point above 9, demonstrating that His176 is the phosphate acceptor in G6Pase. To date, 75 G6Pase mutations have been identified in GSD-Ia patients. These include 48 missense, 9 nonsense, 15 insertion/deletion, and 3 splicing mutations. Interestingly, 64% candidate mutations are missense mutations that result in single amino acid substitutions. Characterization of these mutations will provide valuable information on functionally important residues of the protein. Using site-directed mutagenesis and transient expression assays, we have characterized all 48 missense mutations. The database of residual activity retained by these mutants will serve as a reference in evaluating genotype-phenotype relationships and identifying the minimal G6Pase activity required to correct the GSD-Ia phenotype. G6PT is anchored in the ER by 10-transmembrane helices. To date, 69 G6PT mutations, including 28 missenses and 2 codon-deletions, have been identified in GSD-Ib patients. We previously characterized 15 missense and one codon-deletion mutations using a pSVL-based expression assay. We now report the functional characterization of all 30 codon mutations using an improved G6PT assay based on an adenoviral vector-mediated expression system. Twenty of the naturally occurring mutations completely abolish microsomal G6P uptake activity while the other 10 mutations partially inactivate the transporter. We also report a structure-function analysis of G6PT and show that wild-type and mutant G6PT are degraded in cells predominantly through the proteasome pathway.